Production of recombinant human insulin precursor using Pichia pastoris involves methanol as an inducer for AOX1 promoter and a carbon source. However, the use of methanol is undesirable, especially on a large scale. Alternatively, mixed carbon sources of methanol-glycerol and methanol-sorbitol were examined. The effects on biomass growth and protein expression were analyzed. By using the mixture of methanol-glycerol and methanol-sorbitol, the protein expression levels were comparable to the level produced by using methanol solely. The protein was expressed at several co-substrate compositions despite that glycerol and sorbitol are considered to have repression effects on the AOX1 promoter. It was shown that glycerol and sorbitol could be partial substitutes for methanol in the production of recombinant human insulin precursors using P. pastoris Mut+. Further studies are necessary to evaluate other important parameters before revamping experimental design and verification at larger scale cultivation. [ABSTRACT FROM AUTHOR]